1. Technical Field of the Invention
The present invention relates to an isolated methyl degron peptide and methods of controlling protein lifespan.
2. Description of the Related Art
Ubiquitination is one of the post-translational modifications which regulate not only signalling processes leading to degradation of short-lived regulatory proteins, but also acts as a recognition signal which can activate, or deactivate proteins within a signalling cascade (Pickart, 2004). The covalent attachment of ubiquitin occurs on lysine residues where E1 ubiquitin activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases work in concert (Ciechanover et al., 1982; Hershko, 1983; Hershko et al., 1983). Among the E3 ligase family members, the cullin family, found in vertebrates (e.g., CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and CULT) each allow interaction with specific adapters by acting as a scaffold for ubiquitin ligases (E3). CUL4 family members, including CUL4A and CUL4B, are distinct from other cullins in that they utilize more than 50 WD40-containing adapters, referred to as DCAFs (also known as VprBPs), conferring substrate specificity (Angers et al., 2006; Bennett et al., 2010; He et al., 2006; Higa et al., 2006; Jin et al., 2006).
Apart from ubiquitination, methylation is another post-translational modification that occurs on lysine residues. Among many lysine methyltransferases, Enhancer of Zeste Homolog 2 lysine methyltransferse (EZH2) is a SET domain-containing protein that exhibits histone methyltransferase activity with specificity to histone H3K27 methylation, and forms a polycomb-group repressive complex 2 (PRC2) with EED and SUZ12 (Cao et al., 2002; Caretti et al., 2004; Jones et al., 1998; Kuzmichev et al., 2004; Sewalt et al., 1999). Importantly, EZH2 has been proposed to have oncogenic activity in that it is often deregulated in a number of cancer types (Cao et al., 2011; Chang et al., 2011; Hobert et al., 1996; Kleer et al., 2003; Varambally et al., 2002), and aggressive breast and prostate cancers exhibit high levels of EZH2 which correlates with poor patient prognosis (Fukuyama et al., 2000; Jacobs and van Lohuizen, 2002).
Although nuclear receptors have a variety of different functions to regulate numerous processes by switching transcription on and off via recruiting a complex of co-regulatory proteins (Atkins et al., 1999; Glass and Rosenfeld, 2000), there is increasing evidence that some orphan nuclear receptors play a critical role in tumor suppression. Orphan nuclear receptors are ligand-activated transcription factors, for which no cognate ligands have been identified (Blumberg and Evans, 1998; Giguère, 1999), and one such orphan nuclear receptor includes retinoic acid-related orphan nuclear receptor α (RORα) which acts as an inhibitor of colon cancer growth by trans-repressing canonical Wnt/β-catenin signalling (Lee et al., 2010). DNA damage-induced RORα is involved in the positive regulation of p53 stability leading to increased apoptosis (Kim et al., 2011). RORα has been shown to reduce the migratory and invasive abilities of androgen-independent prostate cancer cells, such as DU145 cells (Moretti et al., 2002). Together, these findings suggest a tumor suppressive role of RORα.
Here, we first provide the evidence that methylation-dependent ubiquitination of RORα is carried out by DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex. We identify DCAF1 as a direct adaptor linking DDB1/CUL4 to RORα by specifically recognizing mono-methylated RORα. Our studies reveal that the chromo domain in DCAF1 functions as a mono-methyl-specific reader by molecular modeling and binding affinity studies. These findings suggest a novel “methyl-degron” pathway where non-histone protein stability is dynamically regulated by methylation. Further, we present physiological data in conjunction with biochemical data strongly supporting the oncogenic role of EZH2 by facilitating RORα methylation-dependent degradation, thereby inhibiting the tumor suppressive role exerted by RORα.